Pseudomonas plecoglossicida attenuated vaccine for fish with T6SS-1 gene cluster knocked out

A kind of Ayu Pseudomonas, T6SS-1 technology, applied in microorganism-based methods, medical preparations containing active ingredients, bacteria and other directions, can solve the problem of the use of commercial vaccines that have not yet been used, and achieve good immunogenicity , Reasonable design and convenient operation

Active Publication Date: 2019-05-31
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the prevention of "visceral white spot disease" of large yellow croaker is mainly based on feeding chemical drugs with ingredients, and there is no commercial vaccine yet.

Method used

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  • Pseudomonas plecoglossicida attenuated vaccine for fish with T6SS-1 gene cluster knocked out
  • Pseudomonas plecoglossicida attenuated vaccine for fish with T6SS-1 gene cluster knocked out
  • Pseudomonas plecoglossicida attenuated vaccine for fish with T6SS-1 gene cluster knocked out

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Construction of T6SS-1 Knockout Strain of Pseudomonas Ayucididae

[0062] Schematic diagram of the process of knocking out the T6SS-1 gene cluster, see figure 2 , firstly, the overlapping PCR approach was used to obtain the spliced ​​fragments of the upstream and downstream sequences of the T6SS-1 gene cluster (the coding gene of T6SS-1 was removed in the middle, and the first three codons of the first gene tssA and the last two codons of the last gene tssM were retained , to ensure that the reading frame does not change), connected to pK18mobSacB (see "Gene", 1994, Vol. In the state Escherichia coli S17-1 / λpir, after copying, proliferating, and extracting the plasmid, carry out PCR and sequencing identification. Specific steps are as follows:

[0063] (1) Overlap PCR: two sets of primers P1 and P2, P3 and P4 were designed using the upstream and downstream sequences of T6SS-1 as templates (the amino acid sequences of which are shown in SEQ ID NO: 1-4, wherein P2 and ...

Embodiment 2

[0112] Comparison of Biological Phenotypes of T6SS-1 Knockout Strain and Wild Type

[0113] The T6SS-1 knockout strain (vaccine strain) and the wild strain were cultured in LB medium, and the growth curve was drawn to compare the difference in growth status between the two. see image 3 , the ecological trend of the T6SS-1 knockout strain was basically synchronized with that of the wild strain, and they both entered the stable phase after 18 hours of cultivation, but the knockout strain A 600 lower value.

Embodiment 3

[0115] Virulence Comparison of Vaccine Strains and Raw Wild Strains

[0116] The virulence of the vaccine strain and the wild strain was evaluated by injecting the original host (large yellow croaker) model. Bacterial cells of the strains in the exponential growth phase were collected separately, and 4 concentration gradients were set up, each of which was 2.0×10 4 , 2.0×10 3 , 2.0×10 2 , 2.0×10 1 CFU / tail, 15 large yellow croakers (body weight 54.3±2.6g) in each group, and a PBS blank control group was set. The bacterial suspensions with adjusted concentrations were quantitatively injected into the large yellow croaker’s abdominal cavity for infection; the infection water temperature was set at 20±2°C, and the observation was continued for 2 weeks after injection, and the death of each animal was recorded, and the statistical model was used to calculate the infection rate of each strain. The median lethal dose (LD50), the results are shown in Table 1. Table 1 shows that ...

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Abstract

The invention provides a pseudomonas plecoglossicida attenuated vaccine for fish with a T6SS-1 gene cluster knocked out, and belongs to the technical field of animal vaccines. The vaccine comprises aknockout strain of one set of VI-secreting system gene clusters of a pseudomonas plecoglossicida genome. A construction method includes adopting an overlapped PCR way to obtain a splicing fragment ofupstream and downstream sequences of the T6SS-1 gene cluster; connecting the splicing fragment to pK18mobSacB to construct a knockout vector and transferring the knockout vector to the competent escherichia coli, transferring the knockout vector to pseudomonas plecoglossicida cells by conjugation and conducting homologous recombination exchange twice to achieve knockout of a target gene. The T6SS-1 gene cluster is knocked out to achieve the pseudomonas plecoglossicida attenuated vaccine with the virulence significantly weakened relative to a wild strain while good immunogenicity is maintained.The vaccine has a good immune protection effect on a host of inoculated fish, and the virulence of the knockout strain does not recover.

Description

technical field [0001] The invention belongs to the technical field of animal vaccines, and in particular relates to an attenuated fish vaccine against Pseudomonas ayucidae for knocking out the T6SS-1 gene cluster. Background technique [0002] Pseudomonas bacteria are widely distributed in soil, fresh water, sea water and organisms. The growth temperature range is wide, and can grow at 4-43 °C. The optimum temperature for most bacteria is around 30 °C, and the optimum pH value for growth In the range of 7.0 to 8.5. Pseudomonas plecoglossicida is a Gram-negative bacterium belonging to the phylum Proteobacteria, the class Gamma-Proteobacteria, the order Pseudomonas, the family Pseudomonas, the genus Pseudomonas, the genus Pseudomonas Pseudomonas group. The bacteria can infect a variety of important economically farmed fish, including sweetfish, large yellow croaker, etc., and it can cause "visceral white spot disease" after infecting large yellow croaker. The disease is pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/78C12N15/66A61K39/104A61P31/04C12R1/38
Inventor 陶震钱冬周素明许文军
Owner ZHEJIANG OCEAN UNIV
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